Development and characterization of microsatellite loci for the primrose Primula vulgaris and successful cross-amplifica
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MICROSATELLITE LETTERS
Development and characterization of microsatellite loci for the primrose Primula vulgaris and successful crossamplification in the congeneric P. elatior and P. veris Miyuki M. Seino • Clara de Vega • Pilar Bazaga Hans Jacquemyn • Carlos M. Herrera
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Received: 3 February 2014 / Accepted: 24 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The perennial herb Primula vulgaris (Primulaceae) faces severe fragmentation of its natural forest habitat in the northern border of its distribution due to intensive agricultural practices and increased urbanisation. In Belgium and northern France, P. vulgaris populations show dramatic declines that could lead to local extinctions. Here we describe 12 novel microsatellite loci obtained by 454-pyrosequencing of a microsatellite-enriched library. Number of alleles per polymorphic locus varied between 2 and 6, and observed heterozygosity ranged from 0.16 to 0.73. Furthermore, sets of 8 loci successfully cross-amplified in the congeneric P. elatior and P. veris. These markers will be of great value for genetic diversity and conservation studies. Keywords 454-pyrosequencing Fragmentation Microsatellites Primula elatior Primula veris Primula vulgaris Primula vulgaris Huds. (=Primula acaulis (L.) L.) is a diploid, self-incompatible (distylous), long-lived perennial herb that flowers in early spring. It has a North-Atlantic and Mediterranean European distribution, growing in humid, shaded habitats in woodlands and open grasslands (Jacquemyn et al. 2009). In west-central Europe, P. vulgaris is a rare, declining species, with highly fragmented
M. M. Seino (&) C. de Vega P. Bazaga C. M. Herrera Estacio´n Biolo´gica de Don˜ana, CSIC, Avda. Ame´rico Vespucio s/n, 41092 Sevilla, Spain e-mail: [email protected] H. Jacquemyn Division of Plant Ecology and Systematics, Biology Department, KU Leuven, Heverlee, Belgium
populations due to habitat destruction as a consequence of urbanisation and intensive agricultural practices (Brys et al. 2004). Here we characterize 12 novel microsatellite loci for this species that, in combination with the 3 loci previously reported by Van Geert et al. (2006), will contribute to gain insights into population genetic diversity and structure which can help to evaluate the species’ conservation status. Most of these microsatellites successfully amplified in the related species Primula elatior (L.) L., which is threatened in the southern margin of its distribution range; and Primula veris L., for which these microsatellites will complement others previously developed by 454 pyrosequencing (Bickler et al. 2013). Genomic DNA was extracted from dried leaf samples using either DNeasy Plant Mini Kit (Qiagen) or Invisorb Spin Plant Mini Kit (Stratec Molecular). Microsatellite loci were isolated by Genoscreen (Lille, France) using a microsatellite enrichment method coupled to 454 GS-FLX Titanium next-generation sequencing (Malausa et al. 2011). Sequences of the oligonucleotide probes used were: TG, TC, AAC, AAG,
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